Kit for detecting Her-2/neu gene by site-specific metal deposition

ABSTRACT

Disclosed are methods and materials for utilizing enzymes to act on metal ions in solution so that the ions are reduced to metal. Additionally disclosed is how to use enzymes to accumulate metal particles. The alteration of metal particles by enzymes interacting with the organic shell of the particles is also described. These methods enable a wide range of applications including sensitive detection of genes and proteins, use as probes for microscopy, nanofabrication, biosensors, and remediation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/822,131, filed Mar. 30, 2001, now U.S. Pat. No. 6,670,113,issued Dec. 30, 2003, the contents of which are incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to novel processes that permit biologicalenzymes to act directly on metals and metal particles. Moreparticularly, one aspect of the invention relates to use of enzymes toselectively deposit metal. Other aspects of the invention relate tolinking of metals to enzyme substrates, control of enzymatic metaldeposition and applications of enzymatic metal deposition.

BACKGROUND OF THE INVENTION

Enzymes: Their Function and Uses

Enzymes are proteins, usually derived from living organisms, that arealso catalysts for various metabolic or chemical reactions. Enzymes aretherefore essential to all life. Recently, enzymes have been isolated,studied, altered, combined with other agents, and used in variousprocesses. Uses of purified enzymes range from laundry detergents (whereenzymes break down stains) to pathological detection of cancer (whereenzymes produce a visible color product on tumor cells in a biopsy).Enzymes can be immobilized, for example by attaching the enzyme to asurface such as a bead, flat surface, or electrode using adsorption orcovalent linkage. Immobilization allows the enzymes to be held in placefor handling or to sustain washing without being removed. Immobilizedenzymes can be used as biosensors, for example to measure glucose levelsfor diabetics.

As previously stated, enzymes are catalysts. As used herein a “catalyst”is defined as a material that increases the rate of a chemical reactionbut is not itself consumed. At the end of a reaction, the catalyst ispresent in its original form so that it may act on new substrates. Asused herein “substrate” is defined as a chemical that an enzyme works onto produce a new chemical. A “substrate” is the input material or“reactant” in the reaction catalyzed by the enzyme. Catalysts functionby binding the substrate chemical or chemicals, and either introducebond strain or orient reactants, thus making a transition or reactionpossible at lower temperature or energy. Since enzymes are catalysts,they lower the activation energy barrier between two chemical states.Enzymes can, for example, facilitate the conversion of one chemicalcompound into another, or facilitate a reaction between chemicals.Without enzymes, reactions would be slow or, for most practicalpurposes, would not occur. This lowering of the activation energybarrier is one reason enzymes are required for living organisms. Enzymescontrol most body processes, and even cancer involves improper levels ofcertain enzymes regulating cell growth and death.

Enzymes fall into various classes relating to the type of reaction theycatalyze, for example: oxido-reductases (such as dehydrogenases,oxidases); hydrolases (such as esterases, lipases, phosphatases,nucleases, carbohydrases, proteases); transferases; phosphorylases;decarboxylases; hydrases; and isomerases. Although enzymes within livingcells act on specific compounds, it has been found that many enzymeswill also act on other related compounds. Enzymes have also been foundto perform similar reactions on synthetic or man-made substrates.

One use of enzymes is to perform reactions that convert a substrate intoa detectable product. For example, a non-fluorescent compound may beconverted into a fluorescent compound by cleavage of a particular bondusing an enzyme. Alternatively, a colorless compound may be convertedinto a colored one by using an enzyme. Other uses of enzymes aredeposition of a colored or otherwise detectable organic substrate fromsolution onto a solid support. This may be done by using an enzyme tomake a soluble starting compound insoluble. Alternatively, enzymes canmake a starting compound reactive, such as by forming a free radicalthereof. The free radical subsequently reacts with, and binds to, thesurrounding material. A useful embodiment of this technology is theELISA test (Enzyme Linked ImmunoSorbant Assay), where, for example, anantigen is adsorbed to a solid support, such as a plastic microtiterplate well. To determine if an antibody to the antigen is present in apatient's serum, the serum is incubated in the coated well. If theantibody is there, it will bind to the immobilized antigen. Afterwashing, a solution containing an anti-human antibody linked to theenzyme alkaline phosphatase is applied. The anti-human antibody willattach to any bound primary antibodies present. After washing, asubstrate is applied, and if the alkaline phosphatase is present it willconvert the colorless BCIP (5-bromo-4-chloro-3-indolyl phosphate) into asoluble color, which can then be measured spectrophotometrically. Theamount of colored product produced is correlated with the amount ofantibody in the serum, providing a quantitative measurement.

A number of significant advantages are gained by using enzymes fordetection. These advantages include: a) Amplification: since the enzymeis a catalyst, and does not get used up in the reaction, and it can beused over and over. As more substrate is added more detectable productis produced. Except for practical limitations, the amount of productproduced could be limitless. b) Linearity: the detectable productproduced from the reaction of enzyme and substrate follows enzymekinetics for that enzyme, and these can be relatively linear within somerange. Even if the particular enzyme kinetics is not linear, thereaction may be calibrated. c) Selectivity: enzymes are usually veryselective for the type of reaction and stereochemistry involved. Thusunwanted interferences may be reduced. d) Low background: if theconversion of the substrate to a colored or otherwise altered compoundis negligible without the enzyme, then the background can be very low.

Enzymes themselves may be modified, for example, by genetic engineeringor chemical modification, to produce alterations in specificity orreactivity, or to impart other characteristics, such as reducedimmunogenicity (if the enzyme is to be used in vivo), or improvedstability (for better shelf life or environmental tolerance).

A further expansion of the enzyme field relates to the use ofunconventional material as biological catalysts, either proteins thatare not normally enzymes, or non-protein material; for example,catalytic antibodies have been described.

Enzyme Substrates for Use in Detection Systems

The types of enzyme substrates popularly used for sensitive detectionare typically calorimetric, radioactive, fluorescent orchemiluminescent. Conventional calorimetric substrates produce a newcolor (or change in spectral absorption) upon enzyme action. This typeof detection is advantageous in that the colors produced are easilydetected by eye or with spectral equipment. The cost of equipment fordetection is also generally less than with other methods; for example inpathology, the brown color produced by the enzyme horseradish peroxidaseacting on 3,3′-diaminobenzidine (DAB), requires only a simple brightfield light microscope for observation of biopsied sections. Adisadvantage of these calorimetric substrates is that they are generallyof lower sensitivity than other enzyme methods.

Conventional radioactive substrates can enzymatically release or fixradioactivity for measurement. Although sensitive, this type ofdetection is becoming less popular due to the risks of handling anddisposing of radioactive material, and since other methods now rival orexceed its sensitivity. Radioactive labeling for histochemical uses andautoradiography, typically require months to expose films, due to lowspecific activity, which is another disadvantage.

Conventional fluorescent substrates are popular since they arereasonably sensitive, generally have low backgrounds, and severaldifferently colored fluorophores can be used simultaneously. A number ofdrawbacks, however, come with use of fluorescent substrates.Fluorescence requires expensive fluorescence optics, light sources andfilters; by comparison, standard bright field microscopes aresignificantly less expensive. Fluorescence fades upon observation,sample storage or even exposure to room lights, thus making permanent orquantitative data difficult to achieve. Autofluorescence (fluorescencecoming from certain compounds found naturally in many living organisms)from cells and other molecules can interfere with the test result.Standard tissue stains (such as nuclear fast red, hematoxylin, andeosin) cannot be seen simultaneously with the very different optics andillumination required for fluorescence detection, thus makingvisualization of landmarks of a tissue difficult. The standard viewingof tissues is done with bright field optics using colored stains and astandard microscope. Unfortunately, fluorescence is viewed usingdifferent illumination and sharp bandpass filters, so that only thefluorescent label is visible, and the general view of the stained tissueis not simultaneously available.

Chemiluminescence is based upon use of substrates that have sufficientlyhigh chemical bond energies so that when the bonds are broken by anenzyme, energy is released in the form of visible light. This method hasgained popularity due to the low background and very high sensitivityobtainable using photomultipliers, avalanche diodes or other sensitivelight detectors. Alternatively photographic film can be used as adetection means. Chemiluminescence has a number of disadvantages.Detection requires expensive equipment or necessitates film development.The sample is not a permanent record, since the emitted light must becollected over time. Sensitive detection often requires lengthy lightintegration times of hours or even a day. Standard stains (such asnuclear fast red, hematoxylin, and eosin) cannot be seen simultaneously,thus making visualization of landmarks of a tissue difficult, forexample. Only the emitted light from points in the specimen can be seen.

Additionally, all of the conventional detection schemes have somepractical limitations for sensitivity. One limiting factor is thebackground, or non-specific signal generated. The background noise cancome from various sources. For example with fluorescence detection thebackground can come from autofluorescence, fluorescent molecules thatadhere to non-specific sites, light reflection off structures and othersources. Another limitation on detection method sensitivity is theamount of signal produced. For example, if an enzyme is used that has alow turnover, and produces only relatively few products, these fewproducts will be harder to detect, and sensitivity will be worse than ifa more efficient enzyme producing more products was used.

As mentioned earlier, since an enzyme is a catalyst and is not used upduring reaction it can be fed more substrate, ideally forming productindefinitely. This provides a form of amplification. Of course there arepractical limitations to enzyme amplification, such as the enzyme losingactivity, the long times necessary to accumulate product, side reactionsor other sources of background limit detection. Further, at some pointthe product produced may interfere with enzyme activity, either byshifting the reaction equilibrium, depositing products so as to impedeflow to the active site of the enzyme or otherwise inhibiting theenzyme.

Although a number of enzyme based assays have been developed, one thatis gaining popularity for sensitive detection is CAtalyzed ReporterDeposition (CARD), also known as Tyramide Signal Amplification (TSA, atrademark of New England Nuclear Corp, subsidiary of Perkin Elmer). Inone variation of this method (there are several variations) abiotinylated antibody or nucleic acid probe detects the presence of atarget by binding thereto. Next a streptavidin-peroxidase conjugate isadded. The streptavidin binds to the biotin. Streptavidin is a proteinisolated from the bacterium Streptomyces. Biotin is an organic compoundhaving the formula C₁₀H₁₆N₂O₃S. A substrate of biotinylated tyramide(tyramine is 4-(2-aminoethyl)phenol) is used which presumably becomes afree radical when interacting with the peroxidase enzyme. The phenolicradical then reacts quickly with the surrounding material, thusdepositing or fixing biotin in the vicinity. This process is repeated byproviding more substrate (biotinylated tyramide) and building up morelocalized biotin. Finally, the “amplified” biotin deposit is detectedwith streptavidin attached to a fluorescent molecule. Alternatively, theamplified biotin deposit can be detected with avidin-peroxidase complex,that is then fed 3,3′-diaminobenzidine to produce a brown color. It hasbeen found that tyramide attached to fluorescent molecules also serve assubstrates for the enzyme, thus simplifying the procedure by eliminatingsteps. Although this type of assay has been used quite successfully, ithas several drawbacks, including: expense of reagents, insufficientamplification, background problems, localization at the ultrastructurallevel (using electron microscopy) can be diffuse, and the limitationsusing fluorophores or chromophores mentioned previously.

Enzyme Biosensors

As used herein a biosensor is a device that uses biological materials tomonitor the presence of a selected material, or materials, in a medium.Enzymes can be used in biosensor applications. Redox(reduction-oxidation) enzymes are used to generate an electrical signal,since electrons are transferred in redox reactions. Various otherenzymes have been used in biosensors, and are selective for thefollowing analytes, including use of beta-glucosidase to detectamygdalin, asparaginase for asparagine, cholesterol oxidase forcholesterol, chymotrypsin for esters, glucose oxidase for glucose,catalase for hydrogen peroxide, lipase for lipids, penicillinase forpenicillin G, trypsin for peptides, amylase for starch, invertase forsucrose, urease for urea, and uricase for uric acid. Generally,biosensors achieve signal transduction using one of three approaches:amperometric, potentiometric and optical.

Amperometric biosensors work by enzymatically generating a currentbetween two electrodes. The simplest design is based on the Clark oxygenelectrode. The Clark oxygen electrode has a platinum cathode and asilver/silver chloride anode. Oxygen is reduced at the platinum cathodeto water, and silver is oxidized to silver chloride at the anode. Therate of electrochemical reaction for the electrode is thereforedependent on the oxygen content of the solution. In a glucose monitor,glucose is a substrate for the immobilized glucose enzyme oxidase, whichoxidizes glucose (consuming oxygen) to produce gluconic acid andhydrogen peroxide. This change in oxygen content alters the electrodecurrent.

A variation on the above method measures the hydrogen peroxide producedby the enzymatic oxidation of glucose by making platinum the anode, andbiasing it to 0.7 volts such that the hydrogen peroxide is oxidized backto oxygen, producing 2 electrons. Although the glucose oxidase isselective for glucose, and does not react with the closely related sugarfructose, some other molecules frequently found in the blood, eitherproducts of normal metabolism (e.g. uric acid) or drugs/medicamentstaken orally (e.g. paracetamol or Vitamin C), can also break downdirectly and electrochemically at the electrode, bypassing the enzymeand giving a spurious signal. Similarly, the enzyme/device interface inother types of known biosensors is often prone to similar non-specificsignals. In another variation redox enzymes may be coupled to otherenzymes that interact with a specific substrate of interest to produce aproduct, the product then driving the redox enzyme.

Potentiometric biosensors are usually based on ion-selective electrodes.Such devices measure the release or consumption of ions during areaction; the simplest potentiometric biosensor is based on a pH-probe.Glucose oxidase, for example, catalyzes the oxidation of glucose togluconate, producing H⁺ ions and hydrogen peroxide. The H⁺ ions are thensensed by the pH probe. Detection is usually in the 10⁻⁴ to 10⁻² Mregion, and therefore the above method generally lacks the accuracy andprecision required for many analytes.

Optical biosensors have two common designs. In a first design lightabsorption is measured. An example is light absorption through a dyehaving a changed color that is the result of an enzyme driven pH change.A second design is based on measuring luminescence. An example is use ofthe enzyme firefly luciferase that reacts with ATP (adenosinetriphosphate) and oxygen to produce AMP (adenosine monophosphate), PP₁(inorganic pyrophosphate), oxyluciferin, CO₂ and a photon of light. Thisreaction can be coupled to any enzyme that produces or consumes ATP.

Metals and Enzymes

Some enzymes contain essential metal ions that are bound and requiredfor activity. The metal ions aid in constraining the substrate for thereaction, but are not themselves consumed or deposited, they are part ofthe catalyst. Examples of enzymes that contain or require metal ions ascofactors are: alcohol dehydrogenase, carbonic anhydrase, andcarboxypeptidase, which all require zinc ions; some phosphohydrolasesand phosphotransferases require magnesium ions, arginase requiresmanganese ions; cytochromes, peroxidase, catalase, and ferredoxincontain iron ions; tyrosinase and cytochrome oxidase contain copperions, pyruvate phosphokinase requires potassium ions, and plasmamembrane ATPase requires sodium ions. However, these metal ions do notserve as substrates, are not linked to substrates and do not deposit asmetal.

A very few metal ions are known to interact with enzyme reactionproducts. For example, the enzyme horseradish peroxidase can produce adiaminobenzidine (DAB) polymer. Nickel or cobalt ions complex with theDAB polymer to give the polymer a darker color. Unfortunately, this usehas not been widely employed since the background goes up substantially,and little improvement in signal-to-noise ratio is generally found.Similarly osmium tetroxide can be added to the DAB polymer after it isformed. The osmium tetroxide reacts with the DAB product and leads toincorporation of the heavy metal, making the DAB deposit more visible inthe electron microscope.

SUMMARY OF THE INVENTION

An object of the invention is to utilize enzymes to accumulate and/ordeposit metal particles.

Another object of the invention is to utilize enzymes to act uponsuitable metal ion substrates resulting in metal deposition.

Yet another object of the invention is to utilize enzymes to act uponsuitable metal ion substrates to produce detectable changes.

Still another object of the invention is to utilize enzymes to act uponsuitable metal ion substrates to produce quantifiable changes.

A further object of the invention to utilize enzymatic reactions thatact on ligands attached to metal particles to produce detectablechanges.

A still further object of the invention to utilize enzymes to act onsubstrates liganded to metal surfaces to produce quantifiable changes.

One embodiment of the present invention describes a method for usingenzymes to selectively catalyze metal deposition. As used herein “metaldeposition” is defined as a buildup or accumulation of metal (metallicelements in the zero oxidation state) in the vicinity of the enzyme.Typically, metal deposition will start within a distance of about 1micron from the enzyme. Naturally as metal deposition continues themetal accumulation may extend beyond this distance. Two aspects aredisclosed. In a first aspect, metal nanoparticles having a diameter inthe range of about 0.8 to 50 nm are linked to, or contain, compoundsthat are acceptable enzyme substrates. When the enzyme deposits thesubstrate compound, the metal particles are co-deposited. In a secondaspect enzymes, or modified enzymes, are used to directly reduce metalions to deposit metal. As used herein “modified enzymes” in thisspecific context are defined to be enzymes that are pretreated with asolution of metal ions before a reducing and oxidizing agent are added.Extensions of the above embodiments included as inventive aspectsinclude electrochemical or electroless plating of the same or adifferent metal over the metal particle or deposit.

In other embodiments of the present invention various applications forusing the enzymatically deposited metals are disclosed. For example, theinventive enzymatically deposited metals may be used for highlysensitive gene detection, sensitive immunodetection, novel biosensordesigns, bacterial detection, remediation and nanofabrication of novelmaterials.

In yet another embodiment of the present invention, a test kit isprovided for enzymatically depositing metal in a zero oxidation state.The test kit comprises means for enzymatically depositing metal in azero oxidation state, and the enzymatically deposited metal provides adetectable test result.

The novel forms described for metals, and/or metal-ligand complexes,participating as substrates for enzymes lead to very distinct advantagesover known methods of detection. Some of these advantages are listedbelow.

No harmful radioactivity need be handled or disposed of to achievecomparable or better sensitivity in assays. No lengthy time of exposureis needed (autoradiograms can take several months to expose). No film,film processing or film chemicals are required.

Gold nanoparticles have extinction coefficients about 1,000 times higherthan highly colored compounds or fluorophores. Therefore, their usesubstantially increases sensitivity.

No expensive fluorescent optics, light sources or filters are required.There is no fading of the sample upon observation or storage or exposureto room lights. Autofluorescence from cells and other molecules does notcreate any interference. Standard stains (such as nuclear fast red,hematoxylin and eosin) can be used and seen simultaneously with theenzyme deposited metals, making visualization of landmarks of a tissuesimple.

No expensive chemiluminescence equipment and/or film development isnecessary. The enzyme deposited metals create a permanent record.Sensitive detection can be relatively quick. No expensivechemiluminescent substrates are needed.

The invention disclosed herein utilizes an enzyme reaction fordeposition or product formation. The deposition rates are determined byenzyme kinetics, which result in linear or easily calibrated rates ofreaction. This makes the invention much more suitable for quantitativedetection than conventional autometallographic methods of depositingmetal on nucleating particles. Additionally the invention does notsuffer from irregular rates of deposition from particle to particle asis known to occur with conventional autometallographic methods.

The present invention has an inherent amplification step since an enzymecatalyst is used. Thus the invention can provide more sensitivedetection than is possible with non-enzymatic methods, such as directimmunolabeling with a fluorophore or chromophore.

Compared with, for example, deposition of an organic molecule; enzymaticmetal deposition makes possible many additional detection methodsincluding by x-rays, electron microscopy, electrochemical, optical,magnetic, and many others. Many of the additional test methods usefulwith enzymatic metal deposits are exceedingly sensitive, and permitassays more sensitive and/or rapid than is possible with conventionaltest methods.

In general, the material of the invention may be alternately formulatedto comprise, consist of, or consist essentially of, any appropriatecomponents herein disclosed. The material of the invention mayadditionally, or alternatively, be formulated so as to be devoid, orsubstantially free, of any components, materials, ingredients, adjuvantsor species used in the prior art compositions or that are otherwise notnecessary to the achievement of the function and/or objectives of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood and objects other than those setforth above will become apparent when consideration is given to thefollowing detailed description thereof. Such description makes referenceto the annexed drawings wherein:

FIG. 1 illustrates the results of a conventional blot test showing colordeveloped by peroxidase acting on DAB (3,3′-diaminobenzidine). Onemicroliter containing one microgram of the enzyme horseradish peroxidasewas applied to a piece of white nitrocellulose membrane and permitted todry. Next, 1 ml of 50 mM TRIS buffer, pH 7.6 was applied containing 10microliters of 10% DAB and 10 microliters of 3% hydrogen peroxide. Notethat when a hydrogen peroxide concentration is used in this application,that concentration is the final or absolute concentration of hydrogenperoxide in the solution. In a few minutes, a brown color appeared overthe area containing the peroxidase (the central spot). However, somebackground is apparent surrounding this spot, as evidenced by the browncolor on the rest of the membrane. The blot was washed with water tostop the reaction. The blot is magnified 7 times.

FIG. 2 illustrates the results of a blot test incorporating one aspectof the invention showing a signal developed by enzyme metal deposition.Similarly to the procedure used in FIG. 1, one microliter containing onemicrogram of the enzyme horseradish peroxidase was applied to a piece ofwhite nitrocellulose membrane and permitted to dry. Next the blot wasincubated with 1 ml of 0.2% silver acetate in water for 3 minutes. Theblot was washed twice with water and a 1 ml solution (comprising 2.5mg/ml hydroquinone, 1 mg/ml silver acetate, and 0.06% hydrogen peroxidein 0.1 M citrate buffer, pH 3.8) was applied. In under a minute, a blackcolored silver metal deposit appeared over the area containing theperoxidase. The blot was washed with water to stop the reaction.Compared to the conventional reaction shown in FIG. 1, the enzyme metaldeposition of this invention shows a much denser and more visibleproduct, and shows no background deposition outside the enzyme area. Theblot is magnified 7 times.

FIG. 3 illustrates the results of a conventional immunostaining testusing an antibody, peroxidase, and the substrate AEC(3-amino-9-ethylcarbazole). Human colon carcinoma tissue was fixed informalin and embedded in paraffin. Next it was sectioned and placed onglass slides for the light microscopy. Sections were deparaffinized withtoluene, and rehydrated through successive washes in 100%, 95%, 80%, 70%ethanol, and finally phosphate buffered saline (PBS: 0.01M sodiumphosphate, 0.14M sodium chloride, pH 7.4). Endogenous peroxide wasquenched by incubating with 2 drops of 3% hydrogen peroxide for 5minutes, followed by washing in PBS. Sections were then incubated with abiotinylated monoclonal antibody to an antigen in the section for 1 hourat room temperature. After washing with PBS, the sections were nextincubated with a streptavidin-peroxidase conjugate for 20 min. Afterwashing with PBS, the substrate AEC was applied in 50 mM acetate buffer,pH 5.0 containing 0.03% hydrogen peroxide. A reddish color developedover certain areas targeted by the monoclonal antibody. The slide waswashed with water to stop the reaction after 20 min. Full width ofbright field light micrograph shown is 250 microns.

FIG. 4. illustrates the results of an immunostaining test incorporatingan aspect of the invention using an antibody, peroxidase, and enzymemetal deposition. Similarly to the procedure used in FIG. 3, human coloncarcinoma tissue was fixed in formalin and embedded in paraffin. Next itwas sectioned and placed on glass slides for the light microscopy.Sections were deparaffinized with toluene, and rehydrated throughsuccessive washes in 100%, 95%, 80%, 70% ethanol, and finally phosphatebuffered saline (PBS: 0.01M sodium phosphate, 0.14M sodium chloride, pH7.4). Endogenous peroxide was quenched by incubating with 2 drops of 3%hydrogen peroxide for 5 minutes, followed by washing in PBS. Sectionswere then incubated with a biotinylated monoclonal antibody to anantigen in the section for 1 hour at room temperature. After washingwith PBS, the sections were next incubated with astreptavidin-peroxidase conjugate for 20 min. After washing with water,the sections were incubated with a solution of 2 mg/ml silver acetate inwater for three minutes, then washed with water. A solution containing2.5 mg/ml hydroquinone and 1 mg/ml silver acetate in a 0.1 M citratebuffer, pH 3.8 was applied, and 3% hydrogen peroxide was added and mixedto achieve a final hydrogen peroxide concentration of 0.06%. Slides wereobserved in the light microscope and metal deposition was stopped with awater wash after 15 min. Intense silver metal staining was observed atthe expected locations. Compared to the conventional immunostainingusing the AEC substrate shown in FIG. 3, the enzyme metal depositionshown here had a similar distribution to the AEC red product, but themetal deposition was more intense, of better resolution, and clearlymore sensitive. Controls where the streptavidin-peroxidase was deletedfrom the procedure showed little background and were clearly negative.Full width of bright field light micrograph shown is 250 microns.

FIG. 5. illustrates the detection of a single gene in a single cellusing the inventive enzyme metal deposition. Silver deposits appear atthe targeted gene site and are evident in the bright field lightmicrograph. This demonstrates the high sensitivity of enzyme metaldeposition to detect single gene copies within individual cells. In themicrograph, the lower cell shows two dense black spots, indicated witharrows, close together, representing the gene on the pair ofchromosomes, as expected, since cells have pairs of chromosomes. In theupper cell, two dense spots, indicated with arrows, are also seen in thecell; they are more widely separated since these cells are ininterphase, and the chromosomes are randomly placed. Full width ofbright field light micrograph is 20 microns.

FIG. 6. illustrates detection of live bacteria using the inventiveenzyme metal deposition. A sample of water microscopically revealedbacterial organisms under phase contrast light microscopy. 1000 parts ofthe water sample was incubated with 1 part of the developing mix(comprising 1 mg/ml silver acetate, 2.5 mg/ml hydroquinone and 0.06%hydrogen peroxide in 0.1 M citrate buffer, pH 3.8). The live bacteriabecame intensely stained black with silver deposits and were easily seenin bright field. The bacteria were actively moving, so were capturedwith a shutter speed of 0.08 sec under bright field conditions using anoil immersion lens at a magnification of 1000×. Most of the bacteria(black spots) were round, or cocci bacteria, but a few rodlike (bacilli)bacteria can be seen in the micrograph. All of the bacteria remainedalive for more than a month. Full width of this micrograph is 100microns.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

One aspect of the invention relates to constructing substrates forenzymes that contain metal particles in the range of about 0.8 to 50 nmin diameter. It should be noted that the terms metal particles and metalclusters are interchangeably used to refer to metal particles. Thisconcept has not been effected nor is it deemed possible by those skilledin the art for several reasons. Enzymes are highly specific; forexample, glucose oxidase will accept glucose and not the closely relatedsugar fructose. Additionally enzymes are stereospecific. Coupling a“large” metal sphere (large in comparison to the substrate, for examplethe metal sphere is sometimes twice the substrate size) to the substratemay alter the substrate structure or present steric hindrances thatwould interfere with enzyme activity. The accepted approach is toenzymatically deposit a small molecule, such as biotin, then detect thiswith streptavidin which is pre-coupled to a gold particle.

Surprisingly, the inventor has discovered that some metalparticle-enzyme substrates, are accepted by some enzymes, and result inspecific deposition of metal particles. One mechanism of metal particledeposition is believed to include oxidation or reduction of thesubstrate forming a species that polymerizes and accumulates creating aninsoluble mass. In another possible mechanism, bonds in the substrateare broken or made which release or alter solubilizing moieties; thesubstrate is converted from a soluble compound into an insolublematerial, which then builds up around the enzyme. In a further possiblemechanism the enzyme creates a free radical on the substrate, making ithighly reactive; the free radical substrate attaches to solid materialaround the enzyme and is covalently fixed in place. Continued enzymeaction adds more product, thus building up a deposit.

This discovery, which as explained above is contrary to both skilledopinions and current practices, has significant advantages. For example,directly depositing metal particles eliminates steps in the existingmethod of biotin deposition followed by streptavidin-gold (or othersimilar processes), thus reducing time and costs. It also brings themetal particles closer to the enzyme for better spatial resolution.Improved spatial resolution is important for electron microscopylocalizations and nanofabrication. Since the metal is depositeddirectly, the efficiency and occupancy of deposition at enzymatic sitesis improved. For electrochemical applications, tunneling and chargeconduction is highly dependent on particle spacing, and the new methodpermits particles to be deposited so that useful conduction may beachieved. Hence, a number of significant benefits are achieved by thisaspect of the invention.

As an example, gold particles in the range of about 0.8 to 50 nm may besynthesized by reduction of gold salts or gold compounds. For example,HAuCl₄ at 0.01% by weight in water is reduced by 2 milliMolar sodiumcitrate in boiling water to produce approximately 10 nm gold particles.By changing the amount of citrate, different sized gold particles may beproduced. Other reducing agents (besides citrate) and recipes are knownfor producing metal particles in the size range of about 0.8 to 50 nm.An organic layer or shell may be bound to the metal particle surface,typically consisting of thiol compounds, phosphines, polymers, proteinsor other compounds. The use of the term organic layer or shell is alsomeant to include partial or incomplete coverage of the metal particlesurface. Some metal particles have these organic coatings attached byadsorption, whereas others are attached by covalent bonding betweensurface metal atoms and, for example, sulfur or phosphorus atoms.

Various methods were found to attach the enzyme substrate to the metalparticle. In one method reactive groups (e.g., amines) of the metalparticle organic layer are used to crosslink to the enzyme substrate.For example, a gold particle stabilized with phosphines containing atleast one aminophoshine may be coupled via the aminophosphine to3,3′-diaminobenzidine. The coupled gold particle-3,3′-diaminobenzidinethen becomes a substrate for horseradish peroxidase, resulting indeposition of gold particle.

In another method the substrate is incorporated directly into the metalparticle organic layer. Gold particles were formed from gold saltsreduced with sodium borohydride in the presence of glutathione and3,3′-diaminobenzidine (DAB), then purified by column chromatography toremove unreacted starting materials. The glutathione and DAB areincorporated into the gold particles. These gold particles were found tobe deposited by horseradish peroxidase in the presence of hydrogenperoxide. Alternatively gold particles can be made from gold saltsreduced with sodium borohydride in the presence of glutathione and4-hydroxythiophenol, then purified by column chromatography to removeunreacted starting materials. The gold particles with incorporatedsubstrate so produced were found to be deposited by horseradishperoxidase in the presence of hydrogen peroxide.

In a further method, gold particles stabilized by thioglucose were foundto be deposited from solution by horseradish peroxidase if hydroquinoneand hydrogen peroxide were included in the solution. These depositedgold particles contained glucose and were also demonstrated to haveactivity with the enzyme glucose oxidase.

Another aspect of the invention is the enzymatic alteration of the metalparticle coating. This aspect of the invention is applicable even if themetal particles are already deposited and remain deposited, or becomesoluble, or if the particles are in solution and remain in solutionafter the enzymatic reaction. For example, a phosphatase will cleave aphosphate group linked to the surface of a gold cluster or particle,thus changing its net charge. The gold particles may remain in solution,but will now move differently in an applied electric field, andtherefore the gold particles will electrophorese differently. Since thegold particles are highly visible and detectable, a sensitive assay ofphosphatase activity may be achieved using this phenomenon.

Another example of a novel enzymatic alteration of a metal particle isthe proteolytic cleavage of a protein or peptide bound to a metalparticle. Upon action of, for example, proteinase K or trypsin, some ofthe protein shell attached to the metal particle was cut by the enzymeand released, resulting in alteration of the absorption spectrum or insome cases aggregation of the metal particles.

Another aspect of the invention is expansion from use with metalparticles to metal surfaces generally. Most of the ligands thatstabilize and bind to metal particles also adhere to metal surfaces. Asused herein “ligand” is defined to be a material that is attached to ametal particle or metal surface. As previously discussed the same, orsimilar, ligands can be used to attach terminal phosphate groups to ametal surface (e.g., using derivatized thiolalkanes or phosphines).Alternatively, the metal particles may be immobilized on a surface. Uponexposure to alkaline phosphatase, the phosphate groups would be cleaved,thus changing the charge of the metal surface. This change can be sensedusing an electrostatic balance or by testing conductivity of thesolution. Thus, metal surfaces are useful platforms for many devices,where enzyme surface alterations can be sensed by optical changes (usingreflectance, or other methods), mass changes (sensed by e.g., a quartzmicrobalance whose frequency shifts with mass), electrical changes(e.g., of conductivity), or other methods.

Metal ions are not believed to be substrates for enzymes. Although someenzymes require metals as part of their active site, the metalsthemselves are not used up, but instead form part of the catalytic sitewith the enzyme to effect other reactions. Most metal ions are cationsand require reduction and electrons for conversion to metal (zerooxidation state). The inventor is not aware of a teaching thatoxidoreductase, or any other, enzymes might be useful to catalyze suchreactions. In fact upon further investigation of the properties ofperoxidases such a reaction would seem unlikely. Horseradish peroxidase,contains as a cofactor ferriprotoporphyrin (a heme group). When bound tothe enzyme, the iron is in the +3 state and linked to a hydroxyl group.This hydroxyl group can be displaced by other anions, including cyanide,azide, fluoride, and its substrate, hydrogen peroxide. The reactionsequence of horseradish peroxidase is proposed to be:a) Enzyme-H₂O+H₂O₂→Enzyme-H₂O₂(complex I)+H₂Ob) Enzyme-H₂O₂+AH₂→complex II+AH.c) complex II+AH.→Enzyme-H₂O+Awhere AH₂ is the reduced substrate and A is the oxidized substrate. Notethat the substrate goes through a free radical form in step b whereinthe free radical is denoted by the dot: “AH.”. As an example,peroxidases catalyze the following reaction:hydroquinone(reduced form)+H₂O₂→quinone(oxidized form)+2H₂OTypically, an organic substrate is oxidized, while hydrogen peroxide isreduced. This is contrary to usage for metal ion reduction since it isdesired to reduce the metal ion, not oxidize it. Furthermore,hydroquinone can reduce some metal ions (depending on concentrations andplace in the electrochemical series, which orders half-reactions byredox potential). However peroxidase would remove hydroquinone fromsolution, instead generating the oxidized form (quinone), which will notreduce metal ions. One would logically conclude that peroxidases weretherefore not suitable for reduction of metal ions.

Contrary to the above conclusion, a surprising and unexpected discoverywas made that under some circumstances, enzymes can accept metal ionsthemselves as a substrate and reduce those metal ions to metal. Furtherthe enzymes can deposit the reduced metal. For example, if horseradishperoxidase is combined with silver ions (silver acetate was usedoriginally), and an appropriate reducing agent is added, e.g.,hydroquinone, no enzyme-mediated reduction of metal occurs. However,upon addition of hydrogen peroxide, the enzyme accepts silver ions as asubstrate and reduces them to silver metal, resulting in a metallicdeposit (a citrate buffer was used at pH 3.8). Therefore another aspectof the invention is enzymatic reduction of metal ions.

It was found that bovine serum albumin and other non-enzyme proteins,for example, IgG, collagen, actin and myosin, when substituted forperoxidase, do not serve to deposit metal. This further confirms thatthe inventive metal deposition is enzymatic in nature.

Further exploration of this phenomenon revealed that pretreatment of theenzyme with gold ions (e.g., from potassium tetrabromoaurate), or silverions (e.g., from silver acetate), followed by optional washing (toremove the excess pretreatment metal ion solution), resulted in greatlyenhanced rates of silver deposition when the developing mix wassubsequently applied. As used herein the term “developing mix” isdefined as the solution applied to the enzyme to obtain metaldeposition. Typically the developing mix contains metal ions (e.g.,silver acetate), a reducing agent (e.g., hydroquinone) and an oxidizingagent (e.g., hydrogen peroxide) in a controlled pH buffer (e.g., 0.1Msodium citrate, pH 3.8). In the above enzymatic metal reduction thedeveloping mix advantageously comprised silver acetate, hydroquinone,and hydrogen peroxide in a citrate buffer at a pH of about 3.8. Onepreferred developing mix consists of 2.5 mg/ml hydroquinone, 1 mg/mlsilver acetate, and 0.06% hydrogen peroxide in 0.1 M citrate buffer, pH3.8. The enzymatic metal reduction and deposition can be convenientlyobserved when the enzyme is immobilized, for example, either onnitrocellulose paper, or immunologically attached to a target antigen.

Although the exact mechanism of this aspect of the invention has notbeen completely elucidated, it may be that hydrogen peroxide is reducedby the enzyme, but some electrons become available to also reduce silvermetal. The hydroquinone is oxidized. It appears that peroxidase or itscofactor may similarly bind the pretreatment metal ions. These boundpretreatment metal ions then either enhance or retard the enzymaticreduction of metal ions supplied in the developing mix. The binding ofmetal ions (e.g., gold or silver) before the developing mix is appliedmay explain the alteration in metal deposition rates seen when thedeveloping mix is applied. Thus, another aspect of the present inventioncomprises alteration of enzyme specificity using a developing mix toallow the enzyme to accept metal ions. A further aspect of the inventionis modulation of the rate of metal deposition by pretreatment of theenzyme.

As discussed above a wide range of silver or gold pretreatmentconcentrations, including about 0.2% silver acetate or about 0.01%potassium tetrabromoaurate, serve to greatly enhance metal deposition.Surprisingly, use of more dilute pretreatment concentrations,particularly including about 0.01% silver acetate is found tosubstantially inhibit the subsequent enzymatic deposition reaction.While inhibition of the enzymatic reaction was unexpected, it provides amethod to retard the enzymatic deposition of metals. Thus, anotheraspect of the invention provides methods to both stimulate and retardthe enzymatic deposition of metals, thereby allowing further control ofthe enzymatic metal deposition process.

Naturally there are variations of the inventive aspects. It has beenfound that some other metal ions may be enzymatically reduced, includingsolutions of mercurous chloride, cesium chloride, lead nitrate, nickelsulfate, copper sulfate, palladium acetate and potassium ferrocyanide.Potassium ferrocyanide was reduced by peroxidase with hydroquinoneadjusted to pH 10.

Other enzymes are also active toward reducing metal ions from theirsalts. For example, with a pretreatment of potassium tetrabromoaurate,catalase was found to reduce silver ions to silver metal whenhydroquinone and hydrogen peroxide were included in a sodium citratebuffer at pH 3.8. Additionally lactoperoxidase was found to be activewith silver ions.

While hydroquinone is presently the best known reducing agent, otherreducing agents are also believed to be useful in practicing theinvention, including, for example, n-propyl gallate, 4-methylaminophenolsulfate, 1,4 phenylenediamine, o-phenylenediamine, chloroquinone,bromoquinone, 2-methoxyhydroquinone, hydrazine,1-phenyl-3-pyrazolidinone (phenidone aminophenol) and dithionite saltssuch as sodium dithionite. 1-phenyl-3-pyrazolidinone, sodium borohydrideand boranes may work as a reducing agent without the need for H₂O₂.

Enzymes may be coupled one after another to produce the desired metaldeposit. For example, glucose serves as a substrate for glucose oxidase,producing hydrogen peroxide. The hydrogen peroxide then serves as asubstrate for peroxidase to deposit silver ions in the presence ofhydroquinone, since hydrogen peroxide is used in that enzyme reaction.

The enzyme altered metal product may be soluble or dispersible in water.Naturally in other embodiments of the invention the altered metalproducts can be soluble in organic solvents. For example,aurothioglucose with hydroquinone and hydrogen peroxide, when exposed tohorseradish peroxidase bound to nitrocellulose, turns an intense yellowat the location of the enzyme, if left undisturbed. However, rinsing oragitation easily disperses the color. This reaction may be coupled to anoptical density reader or used in an ELISA format with a microtiterplate reader that will sense the newly formed colored product.

The inventive metal deposits, formed from particles or ions, may befurther intensified using autometallography. As used herein“autometallography” is defined as a deposition of metal from metal ionsin solution that specifically occurs on a nucleating metal surface. Forexample, it is known that if gold particles in the size range of about 1to 50 nm are exposed to silver ions and a reducing agent, silver metalis deposited on the gold particles forming a composite particle. As moresilver is deposited the composite particle increases in size. As thecomposite particles become larger, they become more visible anddetectable.

Autometallography may be combined with the inventive enzymatic metaldeposition. Once metal has been enzymatically deposited as a metalparticle, the metal particle is subjected to an autometallographicsolution. The autometallographic deposit forms a composite particle andamplifies the size of the enzymatically deposited metal particle so thatthe enzymatic metal deposits are more voluminous and hence easier todetect. The novel combination of enzymatic metal deposition in tandemwith autometallography provides increased sensitivity and/or more rapiddetection.

A further advantage of the use of enzymatic metal deposition in tandemwith autometallography is that the autometallographic deposit may be adifferent metal from that of the enzymatically deposited particle. Thispermits overcoating of the original enzymatic metal deposit by one ormore different autometallographically deposited metal layers. Theautometallographic layer may also become the bulk of the compositeparticle if desired. It should be noted that the autometallographiccoating, or coatings, may confer new properties to the enzymatic metaldeposit, such as altered oxidation rates, magnetic properties, opticalproperties and electrical properties. For example, if gold isautometallographically deposited over an enzymatic silver deposit, thiswould confer improved chemical resistance as gold is more noble and moreresistant to oxidation than the core silver deposit.Autometallographically depositing copper over a enzymatic metal depositwould confer the conductive, and other, properties of copper to themetal particle.

A wide variety of methods can be used to detect and observe the novelenzymatic metal deposits. In fact, the use of an enzymatic metal depositovercomes many of the limitations of conventional enzyme-createdfluorescent or colored particles. Some of the methods useful fordetecting and observing an enzymatic metal deposit include:

Visual observation using the unaided eye. The enzymatic deposition ofsilver or silver after pretreating with gold typically results in ablack or brown color due to the presence of finely divided metal. Thecolor is easily seen by the unaided eye, especially against a lightcolored background.

Visual observation using a microscope. For higher resolution or moresensitive detection, the metal deposit may be viewed under a microscope.The metal is very dense and opaque, and may in many circumstances beeasily detected by this density using bright field illumination.

Reflectance. Since metals reflect light, epi-illumination may be usedeither with or without a microscope. Additionally, metals repolarizelight upon reflection, so crossed polarizers may be used to filter outreflections from non-metallic material, thus improving thesignal-to-noise ratio.

Electron microscopy. Metals are clearly seen via electron microscopy dueto their density in transmission electron microscopy. They also havehigh backscatter coefficients, and may be viewed with a backscatterdetector on a scanning electron microscope. Metals also give offcharacteristic x-rays upon electron bombardment, so they may be detectedby x-ray detectors, or electron energy loss spectrometers. Other methodsinclude detection of the characteristic electron diffraction patterns ofmetals.

Polarographic, electrochemical, or electrical detection. Metalsdeposited on an electrode alter its properties. By probing with theproper currents and voltages, metal can be detected.

X-ray spectroscopy. Metals can be detected by x-ray induced fluorescenceor x-ray absorption.

Chemical tests. Sensitive tests exist for chemically converting metalsinto products that are colored or otherwise detectable.

Mass detection. The mass of the deposited metal is detected using, forexample, a quartz crystal mass balance. A quartz crystal in aninductance-resistance-capacitance (LRC) electronic circuit with analternating voltage supply oscillates at some resonant frequency. Ifmetal is deposited on the surface of the quartz crystal, this changesthe mass of the crystal and its resonant frequency. This provides a verysensitive method for measuring mass changes.

Light scattering. Fine metal deposits will alter the light scattering ofa solution or surface.

Other optical methods of detecting interaction of metals with light,including absorption, polarization and fluorescence.

Magnetic detection. The magnetic properties of the deposited metal canbe detected using appropriate equipment such as magnets, coils, orsensing optical-magnetic property changes.

Autometallography. Further amplification of the signal may be achievedby applying additional metal ions and reducing agent and other additivesto effect further metal deposition that specifically nucleates on theinitial enzymatic metal deposit.

Scanning probe microscopy. Metal deposits may be recognized at highspatial resolution and sensitivity by the various scanning probemicroscope techniques, including scanning tunneling microscopy (STM),atomic force microscopy (AFM), near field optical microscopy (NSOM) andother related techniques using piezoelectrically driven scanned tips.

Many of the enzymes useful in the present invention are purified fromliving cells. Bacteria and living organisms have functioning enzymes.Thus another aspect of the invention is enzymatic metal deposition,under appropriate conditions, in vivo. As one example, an initialdeveloping mix comprising 1 mg/ml silver acetate, 2.5 mg/ml hydroquinoneand 0.06% hydrogen peroxide in 0.1 M citrate buffer, pH 3.8 wasprepared. When the initial developing mix was diluted 1 part developingmix: 1 part solution of a mixture of growing bacteria from pond water,the bacteria stained with silver metal deposits, but rapidly died.

As another example when the initial developing mix was diluted by adding1 part of the above developing mix to 5 parts bacterial solution, thebacteria became black with internal silver deposits in a few minutes,but remained alive.

As a further example when the initial developing mix was diluted byadding 1 part of the above developing mix to 50 parts bacterialsolution, the bacteria also became black with internal silver deposits,but over a longer period of about twenty four hours, and survived withno apparent toxicity. In each of the above examples the bacteria wereintensely stained, much more so than with standard bacterial stains, andeasily seen under a bright field light microscope. The use of enzymaticmetal deposition in vivo provides a novel method of imaging anddetecting bacteria and other live-cells.

The inventive enzymatic metal deposits are useful in a number ofapplications. Thus another aspect of the invention is the use ofenzymatic metal deposits in such applications, including:

Immunohistochemistry and Immunocytochemistry:

Currently, antibodies are widely used to target antigens on cytologicspecimens, tissue slices, and biopsies. The antibodies are then commonlydetected by a variety of techniques including use of a secondaryantibody that is biotinylated, followed by avidin-biotin-peroxidasecomplex (ABC complex), and development of a color with3,3′-diaminobenzidine (DAB). Other detection methods includefluorescence and chemiluminescence. The present invention may be used asa detection scheme by supplying metal ions and a developing mix,preferably with a metal ion pretreatment, to the peroxidase localized tothe antigen by the above described or analogous methods. Instead ofdepositing DAB, a metal, for example silver will be deposited. Enzymaticsilver deposition has the advantage of better detectability not only bybright field microscopy (where a black deposit is formed), but also byreflectance microscopy, electron microscopy and other methods suited tometal detection. Sensitivity is therefore greatly improved overconventional methods.

Alternatively, the inventive metal clusters or colloids with surfaceenzyme substrates described above may be used to form antigen-specificdeposits by enzymatic action. The enzymatic deposition of metals has agreat advantage over simple targeting of metal nanoparticles attached toantibodies, as is commonly done using gold-antibody conjugates forelectron microscopy, lateral flow tests, and other applications. Onemajor advantage is that the metal or metal particles are continuouslydeposited by the enzyme, as long as more substrate is provided. In thisway, huge amounts of metal product may be deposited compared tonon-enzymatic targeting of metal particles, achieving a desirableamplification effect.

Thus, the popular DAB method used widely in pathology may be easilyadapted to the more sensitive enzymatic metal deposition methoddescribed herein to easily achieve better results while requiring fewchanges and little additional expense.

In Situ Hybridization

Presently considerable laboratory and pathological testing is being doneand it is desirable to have a sensitive method to detect DNA or RNAsequences. By hybridizing a complementary probe carrying a detectablemoiety, these sequences can be found and quantified. However the limitsof detection required to see single gene copies or low levels ofexpression exceed the capability of many methods. The present inventionmay be used to achieve sensitive detection by having the nucleic acidprobe labeled with an enzyme, for example peroxidase, or to usemultistep labeling, such as a biotinylated probe, followed byavidin-biotin-peroxidase complex. The peroxidase is then utilized aspreviously described to deposit metals. The enzymatically depositedmetals are highly detectable and as further described below single genesensitivity has been achieved using this novel method.

Lateral Flow, Blots, and Membrane Probing

A number of useful applications, such as lateral flow diagnostics (e.g.,the “dipstick” pregnancy test kit), Western, Southern, and other blots,and other tests performed on membranes, are presently in use. Currently,these methods employ radioactive, fluorescent, colloidal gold,chemiluminescent, calorimetric, and other detection schemes, each havingthe previously discussed drawbacks and limitations. The inventiveenzymatic metal deposition method is readily used with the aboveapplications. For example, the target can be probed with a bindingmoiety that carries an enzyme, for example peroxidase. The bindingmoieties useful depend on the desired target and include, for example,antibody, antibody fragments, antigen, peptide, nucleic acids, nucleicacid probes, carbohydrates, drugs, steroids, natural products fromplants and bacteria and synthetic molecules that have an affinity forbinding particular targets. Enzymatic metal deposition is then applied,using either metal particles with substrate shells or metal ions and anappropriate developing mix, to deposit metal in the vicinity of thespecific target site. The metal deposit may be in the form of anattached deposit or dispersed in solution. The enzymatically depositedmetals are highly detectable and provide an extremely sensitivedetection method.

Sensitive Detection of Antigens and Other Materials

Many other formats for detection of antigens and other materials havebeen devised, such as use of gels, microtiter plate systems and surfacesensors. Most of these may easily be adapted to accommodate the presentinvention and substitute enzymatic deposition of metals and subsequentdetection in place of conventional techniques. This would transform theconventional formats into new and improved formats having desirablecharacteristics such as higher sensitivity, lower cost, permanency ofrecord and other advantages. Substitution of enzymatic deposition ofmetals and subsequent detection in place of conventional techniques alsoeliminates many of the disadvantages of conventional systems, such asuse of radioactive materials, bleaching, transitory products and highexpense.

Electron Microscope Probes

Small amounts of metals are easily detected in electron microscopes bytheir density, backscatter, x-ray emission or energy loss. The inventionherein can be used to specifically target antigens or other sites,initially with an enzyme followed by deposition of metal. Theenzymatically deposited detectable metal allows the targeted sites to beanalyzed with high specificity and sensitivity.

Nanotechnology and Nanofabrication

Since the present invention provides for deposition of metals at enzymelocations, unique materials and objects may be created. For example, astructure such as the regular arrays of muscle tissue may be treatedwith antibodies to the Z band. Then a secondary antibody conjugated toperoxidase is added to the muscle tissue. Finally, the enzyme is given asilver or gold pretreatment, followed by the enzymatic metal deposition.The Z bands are then converted to metal deposits creating ananostructure of finely spaced metallic bands that would be difficult tofabricate by other means. The creation of many other objects andpatterned objects ultimately containing metal using the presentinvention can be imagined by those skilled in the art and all suchembodiments are encompassed by this invention. It should be noted thatenzymatic deposition of magnetic metals will be useful in computertechnology and data storage.

Biosensors

The present invention can be applied to provide a range of novelbiosensors. Several modes of construction are possible, includingamperometric, potentiometric, and optical. For example, an analyte willundergo an enzyme reaction as a substrate. The enzyme reaction mayproduce directly, or be coupled to another enzyme reaction to produce, areducing agent or hydrogen peroxide. As a more specific example glucoseoxidase oxidizes glucose to gluconic acid and hydrogen peroxide. Thusthe enzyme reaction provides the hydrogen peroxide required for thereduction of a metal by peroxidase. The reduced metal is deposited on anelectrode, altering the electrode current. The deposited metal can alsobe sensed polarographically by an alternating current. Potentiometricdetection can be used by sensing the change in metal ion concentrationsresulting from enzyme deposition. Optical biosensors can be made basedon the large light absorption and scattering changes provided byenzymatic metal deposition.

Remediation

Mercury, lead and other toxic metals may be altered by the novel enzymeaction described herein to convert toxic metal ions into insolublemetal, thus fixing the toxic substances and preventing the toxic ionsfrom leeching into groundwater tables. The novel enzymatic action isuseful to remove toxic metals from any water stream, such asmanufacturing or water treatment plants.

Bacteria and Live Cell Staining

The instant invention has been used to intensely stain live bacteria, toa much higher degree than achievable using conventional bacterialstains. This has important implications for detection of bacteria inwater, foods, and samples from patients. Simple metal detectors basedon, for example, light scattering are made possible by the inventiveenzyme metal deposit. Because the enzymatic metal deposit can bedetected with high sensitivity, little or no culturing need be done. Theelimination of the hours or days needed to grow pathogens to adetectable level so that they may be screened for antibiotic sensitivitysolves a problem inherent with current technology. Naturally other livecells and organisms may similarly be stained with novel metaldepositions using enzymes endogenous to that cell or organism.

The present invention also provides a test kit for enzymaticallydepositing metal in a zero oxidation state. The test kit comprises meansfor enzymatically depositing metal in a zero oxidation state, and theenzymatically deposited metal provides a detectable test result.

Having generally described certain aspects of the invention, thefollowing examples are included for purposes of illustration so that theinvention may be more readily understood and are in no way intended tolimit the scope of the invention unless otherwise specificallyindicated.

EXAMPLES Example 1 Enzymatic Deposition of Silver Metal

One microgram of horseradish peroxidase was applied to a nitrocellulosemembrane and allowed to dry. The membrane was then optionally blockedusing 4% bovine serum albumin and washed. A solution containing 2.5mg/ml hydroquinone and 1 mg/ml silver acetate in a 0.1 M citrate buffer,pH 3.8 was applied. Next, hydrogen peroxide was added and mixed to afinal concentration of 0.03 to 0.06%. Silver deposition selectivelyoccurred at the peroxidase spot as evidenced by a black product. Nodeposit occurred if the hydrogen peroxide was omitted.

Example 2 Enhanced Enzymatic Deposition of Silver Metal; Pretreatmentwith Silver Ions

One microgram of horseradish peroxidase was applied to a nitrocellulosemembrane and allowed to dry. The membrane was then optionally blockedusing 4% bovine serum albumin and washed. A solution of 2 mg/ml silveracetate in water was applied for three to five minutes, then washed withwater. A solution containing 2.5 mg/ml hydroquinone and 1 mg/ml silveracetate in a 0.1 M citrate buffer, pH 3.8 was applied. Next, hydrogenperoxide was added and mixed to a final concentration of 0.03 to 0.06%.Silver deposition immediately and selectively occurred at the peroxidasespot as evidenced by an intense black product. No silver depositoccurred if the hydrogen peroxide was omitted.

Example 3 Enhanced Enzymatic Deposition of Silver Metal; Pretreatmentwith Gold Ions

One microgram of horseradish peroxidase was applied to a nitrocellulosemembrane and allowed to dry. The membrane was then optionally blockedusing 4% bovine serum albumin and washed. A solution of 0.1 mg/mlpotassium tetrabromoaurate in water was applied for five minutes, thenbriefly washed with water. A solution containing 2.5 mg/ml hydroquinoneand 1 mg/ml silver acetate in a 0.1 M citrate buffer, pH 3.8 wasapplied. Next, hydrogen peroxide was added and mixed to a finalconcentration of 0.03 to 0.06%. Silver deposition immediately andselectively occurred at the peroxidase spot as evidenced by an intensebrown-black product.

Example 4 Enzymatic Deposition of Gold Nanoparticles

Gold nanoparticles having a diameter in the range of about 1–3 nm and anominal diameter of 1.4 nm were prepared and ligand stabilized with4-hydroxythiophenol and a glutathione such that the 4-hydroxythiophenolwas 30% of the ligand mixture. The gold particles were purified on anAmicon GH25 gel filtration column to remove unreacted materials. Onemicrogram of horseradish peroxidase was applied to a nitrocellulosemembrane and allowed to dry. The membrane was then optionally blockedusing 4% bovine serum albumin and washed. A 10⁻⁶ M solution of thefunctionalized gold nanoparticles in 50 mM Tris buffer, pH 7.6 wasapplied. Next, hydrogen peroxide was added and mixed to a finalconcentration of 0.03 to 0.06%. A brown spot appeared at the peroxidase.

Example 5 Sensitive Immunological Detection of Antigen Using EnzymaticMetal Deposition

Human colon carcinoma resected material was fixed in formalin, paraffinembedded, sectioned, and placed on glass slides for light microscopy.Sections were deparaffinized with toluene, and rehydrated throughsuccessive washes in 100%, 95%, 80%, 70% ethanol, and finally phosphatebuffered saline (PBS: 0.01M sodium phosphate, 0.14M sodium chloride, pH7.4). Endogenous peroxide was quenched by incubating with 2 drops of 3%hydrogen peroxide for 5 minutes, followed by washing in PBS. Sectionswere then incubated with a biotinylated monoclonal antibody to anantigen in the section for 1 hour at room temperature. After washingwith PBS, the sections were next incubated with astreptavidin-peroxidase conjugate for 20 min. After washing with water,the sections were incubated with a solution of 2 mg/ml silver acetate inwater for three minutes, then washed with water. A solution containing2.5 mg/ml hydroquinone and 1 mg/ml silver acetate in a 0.1 M citratebuffer, pH 3.8 was applied, and hydrogen peroxide was added and mixed toa final concentration of 0.06%. Slides were observed in the lightmicroscope and metal deposition was stopped with a water wash after 15min. As shown in FIG. 4, intense silver metal staining was observed atthe antigenic positions detected using conventional means. Parallelslides done using aminoethyl carbinol as the peroxidase substrate showeda similar distribution of red product staining, but it was much lessintense, of poorer resolution and clearly less sensitive than the metaldeposition slides. See FIG. 3. Controls where thestreptavidin-peroxidase was deleted from the procedure showed littlebackground and were clearly negative.

Example 6 Detection of a Single Gene in a Single Cell Using EnzymaticMetal Deposition

With reference to FIG. 5, human breast cancer biopsies were fixed,embedded and sectioned. In situ hybridization was performed by standardprocedures, such as removal of paraffin, treatment with proteinase K,and hybridization with a fluoresceinated probe for the Her-2/neu gene.After hybridization, and washing, a biotinylated anti-fluoresceinantibody was applied, followed by streptavidin conjugated to horseradishperoxidase. Then the sample was pretreated with a gold salt solution,and after washing was exposed to silver acetate, hydroquinone andhydrogen peroxide. Silver deposits appeared at the targeted gene siteand were evident by bright field light microscopy. This demonstrated thehigh sensitivity of the enzyme metal deposition to detect single genecopies within individual cells.

More specifically, the formalin fixed, paraffin embedded and sectionedtissue was deparaffinized by treating with xylene 2×5 min. This wasfollowed by 100% alcohol (2×1 min), 95% alcohol (2×1 min), a water soak(5 min), DakoTarget retrieval solution (40 min at 95° C., then 20 mincool down at room temperature (RT)), a water rinse (5 min with severalchanges), proteinase K (Dako 1:5000 in 50 mM Tris 5 min @ RT), a waterrinse (1×1 min), 80% alcohol (1×1 min), 95% alcohol (1×1 min), 100%alcohol (1×1 min, allowing to air dry), probe addition (10 microlitersof probe, coverslip added and sealed; stored overnight), codenaturation(6 min @ 90° C.), hybridization (37° C. overnight), coverslip soak(remove cement, 2×SSC buffer soak for 5 min), stringent wash (0.5×SSC,72C, 5 min), wash in dd (double distilled) water (2 min), Lugol's iodine(immerse slide for 5 min), dd water (3 rinses), 2.5% sodium thiosulfate(immerse slide for a few sec until tissue clears), dd water (3–5 rinses,7 min total), 1× PBS (phosphate buffered saline)+0.1% Tween 20 (3 minRT), primary antibody incubation (Anti-fluorescein-biotin, 1:100 in PBS,pH 7.6 with 1% BSA (bovine serum albumin), 50 microliters, apply plasticcoverslip, 30 min @ RT), 1×PBS+0.1% Tween 20 (3×5 min, RT), PBS, ph7.6,0.1% fish gelatin (immerse for 5 min), streptavidin-peroxidase (1:200,diluted with PBS pH 7.6 with 1% BSA, 50 microliters, plastic coverslip,60 min @ RT), wash PBS pH 7.6 (2×5 min), PBS pH 7.6, 0.1% fish gelatin(immerse for 5 min), rinse in dd water (10 min several changes), 0.1mg/ml potassium tetrabromoaurate in water (5 min), dd water (3×1 min),substrate silver ions and developing mix (2.5 mg/ml hydroquinone, 1mg/ml silver acetate, 0.06% hydrogen peroxide in 0.1 M citrate buffer,pH 3.8; observe development), stop metal deposition with water wash (2×3min), hematoxylin stain (1 min), water rinse, alcohol-xylene.

Example 7 Enzymatic Deposition of Gold Particles Having3,3′-Diaminobenzidine Attached

Gold nanoparticles having a diameter in the range of about 1–3 nm weresynthesized by reduction of potassium tetrabromaurate with sodiumborohydride, including the thiol ligand glutathione and3,3′-diaminobenzidine. The nanoparticles were then purified fromstarting materials by size exclusion column chromatography on an AmiconGH25 column with water as the eluent. Particles were then incubated withhorseradish peroxidase immobilized on a nitrocellulose membrane, withaddition of hydrogen peroxide to a final concentration of 0.03%. Goldparticles were found to deposit selectively at the peroxidase location.

Example 8 Detection of Live Bacteria Using Enzymatic Metal Deposition

A sample of pond water microscopically revealed bacterial organismsunder phase contrast light microscopy. An aliquot was mixed, 1 partdeveloping mix to 5 parts pond water. The developing mix consisted of 1mg/ml silver acetate, 2.5 mg/ml hydroquinone and 0.06% hydrogen peroxidein 0.1 M citrate buffer, pH 3.8. Over time the bacteria became dark, andafter a few minutes the bacteria were visible by bright field microscopy(whereas they were not seen in this mode previously). The bacteriaremained alive. In another case, 1000 parts tap water was incubated with1 part of the developing mix. After several days, live bacteria wereintensely stained black with silver deposits and were easily seen inbright field. See FIG. 6. The bacteria remained alive for more than amonth.

Example 9 Enzymatic Deposition of Iron

One microgram of horseradish peroxidase was adsorbed to nitrocellulose,dried, then blocked with 4% bovine serum albumin. A solution containing1% potassium ferrocyanide, 0.5% hydroquinone, and 0.06% hydrogenperoxide in a 0.1 M citrate buffer, pH 3.8 was incubated with the targetenzyme. In a few minutes, a deposit of iron was visible as a dense spotat the peroxidase site.

Example 10 Enzymatic Deposition of Mercury

One microgram of horseradish peroxidase was adsorbed to nitrocellulose,dried, then blocked with 4% bovine serum albumin. A solution containing0.5% mercurous chloride, 0.25% hydroquinone, and 0.06% hydrogen peroxidein a 0.1 M citrate buffer, pH 3.8 was incubated with the target enzyme.In a few minutes, a deposit of mercury was visible as a dense spot atthe peroxidase site.

Example 11 Enzymatic Deposition of Nickel

One microgram of horseradish peroxidase was adsorbed to nitrocellulose,dried, then blocked with 4% bovine serum albumin. A solution containing5% nickel sulfate, 0.25% hydroquinone, and 0.06% hydrogen peroxide in a0.1 M citrate buffer, pH 3.8 was incubated with the target enzyme. In afew minutes, a deposit of nickel was visible as a dense spot at theperoxidase site.

Example 12 Enzymatic Deposition of Copper

One microgram of horseradish peroxidase was adsorbed to nitrocellulose,dried, then blocked with 4% bovine serum albumin. A solution containing5% copper sulfate, 0.25% hydroquinone, and 0.06% hydrogen peroxide in a0.1 M citrate buffer, pH 3.8 was incubated with the target enzyme. In afew minutes, a deposit of copper was visible as a dense spot evident atthe peroxidase site.

Example 13 Lactoperoxidase Catalyzed Deposition of Silver

Several beads of immobilized lactoperoxidase were mixed with a solutioncontaining 0.25% hydroquinone, 0.1% silver acetate, and 0.06% hydrogenperoxide in a 0.1 M citrate buffer, pH 3.8. Many of the beads turnedblack in color as a result of silver deposition.

Example 14 Enzymatic Deposition of Silver Using Catalase

One microgram of catalase was adsorbed to nitrocellulose, dried, thenblocked with 4% bovine serum albumin. An aqueous solution containing0.01% potassium tetrabromoaurate was preincubated with the enzyme, thenwashed. Next, a solution of 0.25% hydroquinone, 0.1% silver acetate, and0.06% hydrogen peroxide in a 0.1 M citrate buffer, pH 3.8 was incubatedwith the target enzyme. In a few minutes, a deposit of silver wasvisible as a dense spot at the catalase site.

Example 15 Soluble Colored Compound Containing Gold Formed Upon EnzymeAction on Aurothioglucose

One microgram of horseradish peroxidase was adsorbed to nitrocellulose,dried, then blocked with 4% bovine serum albumin. A solution containing0.5% aurothioglucose was incubated with the enzyme in a solution alsocontaining 0.25% hydroquinone and 0.06% hydrogen peroxide in a 0.1 Mcitrate buffer, pH 3.8. In a few minutes, an intense yellow colored goldproduct was evident at the peroxidase site. The gold product was solubleand could be easily redistributed in the solution if mixed.

Example 16 Enzymatic Deposition of Gold Particles Stabilized withThioglucose

Gold nanoparticles having thioglucose bound to their surfaces wereprepared. The particles were purified by gel filtration chromatographyto remove any excess reactants. One microgram of horseradish peroxidasewas adsorbed to nitrocellulose, dried, then blocked with 4% bovine serumalbumin. A solution containing 0.5 O.D. at 420 nm of the gold particleswas incubated with the enzyme in a solution also containing 0.25%hydroquinone and 0.06% hydrogen peroxide in a 0.1 M citrate buffer, pH3.8. In a few minutes, an brown product was evident at the peroxidasesite, indicating deposition of the nanoparticles.

Example 17 Sensitive Immunological Detection Using Enzymatic MetalDeposition

Human breast biopsy material was prepared in a manner similar to Example5. Four tests were done on the biopsy material using the inventiveenzymatic metal deposition and conventional (DAB) staining techniques:

1. breast cancer, testing for and localizing the estrogen receptor (ER);

2. breast cancer, testing for and localizing the progesterone receptor(PR);

3. breast cancer, testing for and localizing the Her 2-neu oncoprotein;and

4. breast cancer, testing for and localizing the Her 2-neu oncoprotein.

Slides were observed in the light microscope and metal deposition wasstopped with a water wash after 15 min. The samples prepared using theinventive enzymatic metal deposition method had deposits of much higherresolution than the conventionally stained test samples, so that, forexample, membrane proteins were clearly localized to the membrane. Themetal deposit signal was very intense and black with a higher densitythan the conventionally stained test samples. There was no backgroundusing the inventive method. Parallel slides done using DAB resulted in abrown signal that was less dense than the signal produced using theinventive method.

Example 18 Enzymatic Deposition of Metal Using 4-MethylaminophenolSulfate as the Reducing Agent

One microliter of a 0.1 mg/ml solution of horseradish peroxidase wasapplied to nitrocellulose paper and allowed to dry. 50 microliters of a2 mg/ml solution of silver acetate was applied, followed by addition of50 microliters of 0.1 M sodium citrate buffer, pH 3.4. Next, 5microliters of a 5 mg/ml solution of 4-methylaminophenol sulfate (alsocalled metol or elon) was applied with mixing. An identical test wasprepared except that additionally 3 microliters of a 3% hydrogenperoxide solution was added with mixing. Only the sample with thehydrogen peroxide showed specific deposition of silver metal at thehorseradish peroxide site on the nitrocellulose, as evidenced by a darkmetallic spot congruent with the position of the bound enzyme.

Example 19 Enzymatic Deposition of Metal Using 1,4 Phenylenediamine asthe Reducing Agent

One microliter of a 0.1 mg/ml solution of horseradish peroxidase wasapplied to nitrocellulose paper and allowed to dry. 50 microliters of a2 mg/ml solution of silver acetate was applied, followed by addition of50 microliters of 0.1 M sodium citrate buffer, pH 3.4. Next, 5microliters of a 5 mg/ml solution of 1,4 phenylenediamine was appliedwith mixing. An identical test was prepared except that additionally 3microliters of a 3% hydrogen peroxide solution was added with mixing.Only the sample with the hydrogen peroxide showed specific deposition ofsilver metal at the horseradish peroxide site on the nitrocellulose, asevidenced by a dark metallic spot congruent with the position of thebound enzyme.

Example 20 Enzymatic Deposition of Metal Using O-Phenylenediamine as theReducing Agent

One microliter of a 0.1 mg/ml solution of horseradish peroxidase wasapplied to nitrocellulose paper and allowed to dry. 50 microliters of a2 mg/ml solution of silver acetate was applied, followed by addition of50 microliters of 0.1 M sodium citrate buffer, pH 3.4. Next, 5microliters of a 5 mg/ml solution of o-phenylenediamine was applied withmixing. An identical test was prepared except that additionally 3microliters of a 3% hydrogen peroxide solution was added with mixing.Only the sample with the hydrogen peroxide showed specific deposition ofsilver metal at the horseradish peroxide site on the nitrocellulose, asevidenced by a dark metallic spot congruent with the position of thebound enzyme.

Example 21 Enzymatic Deposition of Metal Using Chloroquinone as theReducing Agent

One microliter of a 0.1 mg/ml solution of horseradish peroxidase wasapplied to nitrocellulose paper and allowed to dry. 50 microliters of a2 mg/ml solution of silver acetate was applied, followed by addition of50 microliters of 0.1 M sodium citrate buffer, pH 3.4. Next, 5microliters of a 5 mg/ml solution of chloroquinone was applied withmixing. An identical test was prepared except that additionally 3microliters of a 3% hydrogen peroxide solution was added with mixing.Only the sample with the hydrogen peroxide showed specific deposition ofsilver metal at the horseradish peroxide site on the nitrocellulose, asevidenced by a dark metallic spot congruent with the position of thebound enzyme.

Example 22 Enzymatic Deposition of Metal Using Bromoquinone as theReducing Agent

One microliter of a 0.1 mg/ml solution of horseradish peroxidase wasapplied to nitrocellulose paper and allowed to dry. 50 microliters of a2 mg/ml solution of silver acetate was applied, followed by addition of50 microliters of 0.1 M sodium citrate buffer, pH 3.4. Next, 5microliters of a 5 mg/ml solution of bromoquinone was applied withmixing. An identical test was prepared except that additionally 3microliters of a 3% hydrogen peroxide solution was added with mixing.Only the sample with the hydrogen peroxide showed specific deposition ofsilver metal at the horseradish peroxide site on the nitrocellulose, asevidenced by a dark metallic spot congruent with the position of thebound enzyme.

Example 23 Enzymatic Deposition of Metal Using 2-Methoxyhydroquinone asthe Reducing Agent

One microliter of a 0.1 mg/ml solution of horseradish peroxidase wasapplied to nitrocellulose paper and allowed to dry. 50 microliters of a2 mg/ml solution of silver acetate was applied, followed by addition of50 microliters of 0.1 M sodium citrate buffer, pH 3.4. Next, 5microliters of a 5 mg/ml solution of 2-methoxyhydroquinone was appliedwith mixing. An identical test was prepared except that additionally 3microliters of a 3% hydrogen peroxide solution was added with mixing.Only the sample with the hydrogen peroxide showed specific deposition ofsilver metal at the horseradish peroxide site on the nitrocellulose, asevidenced by a dark metallic spot congruent with the position of thebound enzyme.

Example 24 Enzymatic Deposition of Metal Using Hydrazine as the ReducingAgent

One microliter of a 0.1 mg/ml solution of horseradish peroxidase wasapplied to nitrocellulose paper and allowed to dry. 50 microliters of a2 mg/ml solution of silver acetate was applied, followed by addition of50 microliters of 0.1 M sodium citrate buffer, pH 3.4. Next, 5microliters of hydrazine was applied with mixing. An identical test wasprepared except that additionally 3 microliters of a 3% hydrogenperoxide solution was added with mixing. Only the sample with thehydrogen peroxide showed specific deposition of silver metal at thehorseradish peroxide site on the nitrocellulose, as evidenced by a darkmetallic spot congruent with the position of the bound enzyme.

Example 25 Enzymatic Deposition of Metal Using Dithionite Salt (e.g.,Sodium Dithionite) as the Reducing Agent

One microliter of a 0.1 mg/ml solution of horseradish peroxidase wasapplied to nitrocellulose paper and allowed to dry. 50 microliters of a2 mg/ml solution of silver acetate was applied, followed by addition of50 microliters of 0.1 M sodium citrate buffer, pH 3.4. Next, 5microliters of a 5 mg/ml solution of sodium dithionite was applied withmixing. An identical test was prepared except that additionally 3microliters of a 3% hydrogen peroxide solution was added with mixing.Only the sample with the hydrogen peroxide showed specific deposition ofsilver metal at the horseradish peroxide site on the nitrocellulose, asevidenced by a dark metallic spot congruent with the position of thebound enzyme.

Example 26 Enzymatic Deposition of Metal Using Thioglucose as theReducing Agent

One microliter of a 0.1 mg/ml solution of horseradish peroxidase wasapplied to nitrocellulose paper and allowed to dry. 50 microliters of a2 mg/ml solution of silver acetate was applied, followed by addition of50 microliters of 0.1 M sodium citrate buffer, pH 3.4. Next, 5microliters of a 100 mg/ml solution of thioglucose was applied withmixing. An identical test was prepared except that additionally 3microliters of a 3% hydrogen peroxide solution was added with mixing.Only the sample with the hydrogen peroxide showed specific deposition ofsilver metal at the horseradish peroxide site on the nitrocellulose, asevidenced by a dark metallic spot congruent with the position of thebound enzyme.

Example 27 Enzymatic Deposition of Metal Using Glucosamine as theReducing Agent

One microliter of a 0.1 mg/ml solution of horseradish peroxidase wasapplied to nitrocellulose paper and allowed to dry. 50 microliters of a2 mg/ml solution of silver acetate was applied, followed by addition of50 microliters of 0.1 M sodium citrate buffer, pH 3.4. Next, 20microliters of a 5 mg/ml solution of glucosamine was applied withmixing. An identical test was prepared except that additionally 3microliters of a 3% hydrogen peroxide solution was added with mixing.Only the sample with the hydrogen peroxide showed specific deposition ofsilver metal at the horseradish peroxide site on the nitrocellulose, asevidenced by a dark metallic spot congruent with the position of thebound enzyme.

Example 28 Enzymatic Deposition of Metal Using Sodium Metabisulfite asthe Reducing Agent

One microliter of a 0.1 mg/ml solution of horseradish peroxidase wasapplied to nitrocellulose paper and allowed to dry. 50 microliters of a2 mg/ml solution of silver acetate was applied, followed by addition of50 microliters of 0.1 M sodium citrate buffer, pH 3.4. Next, 5microliters of a 5 mg/ml solution of sodium metabisulfite was appliedwith mixing. An identical test was prepared except that additionally 3microliters of a 3% hydrogen peroxide solution was added with mixing.Only the sample with the hydrogen peroxide showed specific deposition ofsilver metal at the horseradish peroxide site on the nitrocellulose, asevidenced by a dark metallic spot congruent with the position of thebound enzyme.

Example 29 Enzymatic Deposition of Metal Using Aminophenol as theReducing Agent

One microliter of a 0.1 mg/ml solution of horseradish peroxidase wasapplied to nitrocellulose paper and allowed to dry. 50 microliters of a2 mg/ml solution of silver acetate was applied, followed by addition of50 microliters of 0.1 M sodium citrate buffer, pH 3.4. Next, 5microliters of a 5 mg/ml solution of aminophenol was applied withmixing. An identical test was prepared except that additionally 3microliters of a 3% hydrogen peroxide solution was added with mixing.The sample with the hydrogen peroxide showed specific deposition ofsilver metal at the horseradish peroxide site on the nitrocellulose, asevidenced by a dark metallic spot congruent with the position of thebound enzyme. A spot over the enzyme also developed in the samplewithout hydrogen peroxide, indicating that the hydrogen peroxide may notbe required in this case.

Example 30 Enzymatic Deposition of Metal Using 1-Phenyl-3-Pyrazolidinone(Phenidone) as the Reducing Agent

One microliter of a 0.1 mg/ml solution of horseradish peroxidase wasapplied to nitrocellulose paper and allowed to dry. 50 microliters of a2 mg/ml solution of silver acetate was applied, followed by addition of50 microliters of 0.1 M sodium citrate buffer, pH 3.4. Next, 5microliters of a 5 mg/ml solution of 1-phenyl-3-pyrazolidinone I wasapplied with mixing. An identical test was prepared except thatadditionally 3 microliters of a 3% hydrogen peroxide solution was addedwith mixing. The sample with the hydrogen peroxide showed specificdeposition of silver metal at the horseradish peroxide site on thenitrocellulose, as evidenced by a dark metallic spot congruent with theposition of the bound enzyme. A spot over the enzyme also developed inthe sample without hydrogen peroxide, indicating that the hydrogenperoxide may not be required in this case.

Example 31 Enzymatic Deposition of Metal Using Borohydride as theReducing Agent

One microliter of a 0.1 mg/ml solution of horseradish peroxidase wasapplied to nitrocellulose paper and allowed to dry. 50 microliters of a2 mg/ml solution of silver acetate was applied, followed by addition of50 microliters of 0.1 M sodium citrate buffer, pH 8. Next, 5 microlitersof a 1 mg/ml solution of sodium borohydride was applied with mixing. Anidentical test was prepared except that additionally 3 microliters of a3% hydrogen peroxide solution was added with mixing. The sample with thehydrogen peroxide showed specific deposition of silver metal at thehorseradish peroxide site on the nitrocellulose, as evidenced by a darkmetallic spot congruent with the position of the bound enzyme. A spotover the enzyme also developed in the sample without hydrogen peroxide,indicating that the hydrogen peroxide may not be required in this case.

Those skilled in the art will recognize, or be able to ascertain with nomore than routine experimentation, many equivalents to the specificembodiments of the invention disclosed herein. Such equivalents areintended to be encompassed by the scope of the invention.

1. A kit for detecting Her-2/neu gene or protein in a test sample,comprising: metal ions selected from the group consisting of silver,gold, iron, mercury, nickel, copper, platinum, palladium, cobalt,iridium ions and a mixture thereof; an oxidizing agent; a reducingagent; an enzyme; and a binding moiety that binds to Her-2/neu gene orprotein in the test sample.
 2. The kit of claim 1, wherein the bindingmoiety is a nucleic acid probe predetermined to bind to Her-2/neu gene.3. The kit of claim 2, wherein the nucleic acid probe is labeled withbiotin.
 4. The kit of claim 1, wherein the binding moiety is a primaryantibody predetermined to bind to Her-2/neu protein.
 5. The kit of claim4, wherein the enzyme is conjugated to a secondary antibody that bindsto the primary antibody.
 6. The kit of claim 1, wherein the enzyme isperoxidase; the metal ions are silver ion; the oxidizing agent ishydrogen peroxide; and the reducing agent is hydroquinone.